Gel filtration of nucleic acids on pearl-condensed agarose

Vol. 20, No. 1,1965

BKKHEMICAL AND BKMIYSICAL RESEARCH COMMUNICATIONS

GEL FILTRATION OF NUCLEIC ACIDS ON PEARL-CONDENSED AGAROSE 1 B. Gberg, S. Bengtsson Division

of Cell

and Institute

Biology,

and L. Philipson

Department

of Biochemistry,

of Medical

University

Microbiology

of Uppsala,

Uppsala,

Sweden ReceivedMay17,

1965

The use of gel filtration

to separate

2'

by the introduction

lo5 was made possible

(1961).

These gels might allow

ding to size.

An artificial

on granulated

1.5% agarose

in gels with this

molecules

separation

mixture

was overcome by the introduction

The present poliovirus sition

study

describes

Agarose

preparation.

Pearl-condensing. according sieved

et al.

2% agarose.

The low flow

rates

to use but

agar or agarose

acids

(Bengts-

from KS cells

is also shown that

pattern

of poliovirus

from Special

(1964).

in water

and

the compoRNA.

agar Noble (Difco)

by

type 1 (strain

ted with

phenol as described

previously

labelled

with

was pearl-condensed

The pearl-condensed

60 - 100 mesh (US.sieve

RNA. Poliovirus

l

It

of 2% agarose

Poliovirus

32P, since

accor-

(1964).

and Philipson

and the fraction

1962).

of nucleic

the elution

A hot solution

to Bengtsson

acids

RNA has been separated

of pearl-condensed

Agarose was prepared

the method of Russel

nucleic

than 3% made them difficult

gel filtration

influences

above

1964).

RNA on pearl-condensed

of the buffer

weights

agar gels by Polson

of high molecular

of T2 DNA and E. coli

less

1964, Hje&n,

molecular

of granulated

gel (Boman and Hjert&,

agar concentrations

son and Philipson,

with

series)

was collected.

~206) was grown, (ijberg

the amount of poliovirus

et al.,

agarose was

purified

1965).

and extrac-

The virus

was

RNA was too small to be mea-

This work was supported by grants from Damon Runyon Memorial Fund, Jane Coffin Child5 Memorial Fund and the Swedish Medical Research Council (Y 420).

36

BlOCHEMlCAL AND BIOPHYSKAL RESEARCH COMMUNICATIONS

Vol. 20, No. 1,1965

sured by W-absorbancy. KH-nucleic

acids.

KH cells

ner medium (Eagle, centrifugation.

were grown in spinner

1959) with

The nucleic

double strength

method (Philipson,

Gel filtration.

A glass-column,

the column. buffers

a suspension

ments were performed fractions

Fig.

of pearl-condensed

to prevent

at room temperature

of 2 ml were collected.

was applied

1.

2% agarose

to sediment

bacterial

before

contamination.

a flow

rate

disc

was in

use. All The experi-

of 2 ml/cm2/hour

and

acid preparation

in each experiment.

Gel filtration of poliovirus RNA labelled with 32P on a 2% agarose cohun~~, 1.8 x 26 cm, $t room temperature in different buffers, The flow rate was 2-p/cm /hour and 2 ml fractions were collected. The buffers2were 10 M lithium phosphate, pH 6.0 (_ogen circles) and 2 : 10 M lithium phosphate pH 6.0 with 2 * 10 M MgS04 (filled circles) Fig.

agarose

gel column with

the same poliovirus

eluting

RNA, which

buffers.

1 shows two experiments

In 10-3 M lithium

has a molecular

weight

lume and is thus not retarded. MgS04 the RNA eluted

probably

by

by the

glass

About 1 ml of the nucleic

and discussion.

10-3M

pellet

a sintered

500 ml of buffer

with

Results

ferent

and harvested

from the cell

1.8 x 45 cm, with

0,5$ butanol

MEM spin-

1961).

The column was then washed with

contained

in Eagle's

of amino acids

acids were extracted

phenol-duponol

packed by allowing

cultures

the tendency

performed

RNA preparation, phosphate

buffer,

of about 2 x 106, elutes

In 1G-2M lithium

phosphate

at 60% of the bed volume.

of RNAto assumedifferent

37

on the same 2% but with

two dif-

pH 6.0 poliovirus with

the void vo-

buffer

pH 6.0 with

The reason

for

this

is

conformation in the two buf-

Vol. 20, No. 1,1965

BIOCHEMICAL

fers. According to Spirin lutions

of relatively

AND BlOPHYSlCAL

RESEARCH COMMUNICATIONS

(1964) high molecular weight RNAforms rods in so-

low ionic strength at room temperature. These rods are

transformed to more compact coils in higher ionic strength and also in the presence of divalent

metal ions as magnesium.Since gel filtration

molecules of different variation

size, but not necessarily

of different

in ionic strength and composition of the buffer

molecular weight,

gives a possibility

to separate nucleic acids of the same size, but with different form coils or rods. It ded helical

should therefore

separates

tendency to

be possible to separate a double stran-

form of DNAor RNAfrom a single stranded form with the samemole-

cular weight, the single strand beeing more easily coiled in a compact form, when raising the ionic strength or adding magnesiumions. Preliminary with double stranded poliovirus

RNAsupport this hypothesis.

!Cherecovery in experiments with poliovirus sured as radioactivity,

results

RNAwas usually around loo%, mea-

but in someexperiments all RNAwas adsorbed to the

column. The reason for this is not clear,

hut since the amount of poliovirus

RNAapplied to the column was in the order of 0.05 ug, somecharged groups in the agarose could be responsible for the adsorption. When poliovirus with KB-nucleic acids was chromatographed a quantitative

RNAmixed

recovery was always

obtained. A mixture of K&nucleic

acids and poliovirus

RNAwas filtered

of 2% agarose using 2 ' 10'3M sodium phosphate buffer as eluant.

pH 6.0 with 10-3bJlMgCl2

As shown in Fig, 2 the KR-nucleic acids were separated into three

classes. The first transfer

through a column

UV peak is DNA, the second ribosomal RNA's and the third

RNA's as determined by the diphenylamine reaction

the orcinol

reaction

centrifugation.

for RNA (Kerr and Seraidarian,

for DNA (Rurton,l956),

1945) and analytical

ultra-

The ribosomal RNA's are thus not separated in two classes as

might be expected. The reason for this is unclear, but the degree of coiling might be different

in the two classes. Poliovirus

RNA elutes between DNAand the

ribosomal RNA's. The recovery is 80 - 1005, counted as radioactivity sorption.

The position

of poliovirus

RNAindicates

38

and UV ab-

that high molecular RNA's

Vol. 20, No. 1, 1965

BlOCHEMlCAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Per

cent

of bed

volume

Fig. 2. Gel filtration of a mixture of poliovirus RNA labelled with 32 P and KBnucleic acids, on a 23 agarose column, 1.8 x 40 cm, at room temperature. The flow ray was 2 ml/cm /hour and 2 ml fracijons were collected. The buffer was 2 10 M sodium phosphate pH 6.0 with 10 M MgCl,.

could be separated from each other. To achieve a complete separation a longer column would be necessary. Experiments on pearl-condensed gels with varying agarose concentrations

have shown the separation effect

of 1% agarose gels to

be superior to that of 2% gels for nucleic acids with molecular weights above 5

l

105. On 4% gels DNAand ribosomsl RNA's elute together in the void volume,

but the transfer

RNA's are well separated from the other nucleic acids. As poin-

ted out above the separation is highly dependent on the buffer conclude it has been shown that gel filtration provides an additional

composition. To

on pearl-condensed agarose gels

tool for separation of different

classes of nucleic acids.

REFERENCES Bengtsson, S. and Philipson, L., Biochim.Biophys. Acta, 2, 3gg (1964). Boman, H. and Hjertin, S., Arch.Biochem.Biophys., suppl. 1, 276 (1962). Burton, K., Biochem. J., 62, 315, (1956). Eagle, If., Science, 130, 432,(1959). Hjert'en, S., Biochim. Biophys. Acta, 2, 393 (1964).

39

Vol. 20, No. 1,1965

Kerr,

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMJNlCAllONS

S.E. and Seraidairan,

K., J.Biol,

Chem., 2,

211 (1945).

Philipson, L., J. Gen. Physiol., 11, 899 (1961). Polson, A., Biochim. Biophys. Acta, 2, 565 (1961). Russell, Spirin,

B., Mead, T.H. and Polson, A., Biochim. Biophys, Acta, 86, 169, (1964). A.S., Macromolecular Structure of Ribonucleic Acids, Reinhold

Publishing Corporation, Gberg, B., Albertsson,

New York, 1964. P.-A, and Philipson,

(w3).

40

L.,

Biochim.

Biophys.

Acta,

in press,